MCP-1 Antibody Treatment Enhances Damage
and Impedes Repair of the Alveolar Epithelium
in Influenza Pneumonitis
T. Narasaraju1, H. H. Ng1, M. C. Phoon1, and Vincent T. K. Chow1
1
Infectious Diseases Program, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore,
Kent Ridge, Singapore
Recent studies have demonstrated an essential role of alveolar
macrophages during influenza virus infection. Enhanced mortalities
were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis
is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis
resulted in enhanced alveolar epithelial damage and apoptosis, as
evaluated by histopathology, immunohistochemistry, Western blot,
RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment
was achieved by treatment with monoclonal antibody against
monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus.
Interestingly, elevated levels of hepatocyte growth factor (HGF),
a mitogen for alveolar epithelium, were detected in bronchoalveolar
lavage samples and in lung homogenates of control untreated and
nonimmune immunoglobulin (Ig)G-treated mice after infection
compared with anti–MCP-1–treated infected mice. The lungs of
control animals also displayed strongly positive HGF staining in
alveolar macrophages as well as alveolar epithelial cell hyperplasia.
Co-culture of influenza virus–infected alveolar epithelial cells with
freshly isolated alveolar macrophages induced HGF production and
phagocytic activity of macrophages. Recombinant HGF added to
mouse lung explants after influenza virus infection resulted in
enhanced BrdU labeling of alveolar type II epithelial cells, indicating
their proliferation, in contrast with anti-HGF treatment showing
significantly reduced epithelial regeneration. Our data indicate that
inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that
HGF produced by macrophages in response to influenza participates
in the resolution of alveolar epithelium.
Keywords: alveolar epithelial regeneration; apoptosis; hepatocyte
growth factor; influenza pneumonitis; Monocyte chemoattractant
protein-1
Influenza virus is enveloped with a segmented negative-sense
RNA genome, and belongs to the Orthomyxoviridae family. In
recent years, frequent outbreaks of influenza caused by the
H5N1 subtype have resulted in fatalities in humans, poultry, and
other animal species worldwide (1, 2). The global circulation of
H3N2 virus strains is significant, and there is evidence for
genetic compatibility of reassortants derived from H5N1 and
H3N2 viruses (3, 4). Recently, the novel swine-origin influenza
(Received in original form November 3, 2008 and in final form June 26, 2009)
This study was supported by research grants from the National Medical Research
Council, Singapore and the Microbiology Vaccine Initiative, National University
of Singapore (to V.T.K.C.).
Correspondence and requests for reprints should be addressed to Vincent Chow,
M.D., Ph.D., Infectious Diseases Program, Department of Microbiology, Yong
Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2,
Kent Ridge 117597, Singapore. E-mail: [email protected]
Am J Respir Cell Mol Biol Vol 42. pp 732–743, 2010
Originally Published in Press as DOI: 10.1165/rcmb.2008-0423OC on July 17, 2009
Internet address: www.atsjournals.org
CLINICAL RELEVANCE
This study demonstrated that inhibition of macrophage
recruitment augmented alveolar epithelial damage and
apoptosis in influenza virus infection, suggesting the protective role of alveolar macrophages in the resolution of
alveolar epithelium via hepatocyte growth factor (HGF)
production. Our findings indicate the active role of HGF in
the regeneration and resolution of alveolar epithelium
after influenza virus infection.
A H1N1 virus is spread to many countries around the world,
leading to a pandemic (5). The epithelial lining of the respiratory tract including nasal, tracheal, bronchial, and alveolar
epithelia are targets for influenza virus replication (6–8). Influenza viruses also infect monocytes, macrophages, and other
leukocytes (9, 10).
The accumulation of macrophages has been linked with
immunopathology during influenza virus infection, as they
produce proinflammatory cytokines (e.g., TNF-a, IL-1, IL-6,
GM-CSF), chemokines (including IP-10, MIP-1a, RANTES),
and also stimulate expression of inducible nitric oxide synthase
(9, 11–13). Chemokines are small peptide molecules with
chemotactic and activating effects on leukocytes. Among chemokines, monocyte chemoattractant protein-1 (MCP-1) is the
major chemoattractant responsible for the recruitment of
macrophages, but also attracts neutrophils and T-lymphocytes
(14–16). Mice lacking MCP-1 display decreased macrophage
and neutrophil infiltration with increased viral load and elevated levels of TNF-a, IL-6, MIP-2, and IFN-g, suggesting that
MCP-1 contributes to an adequate protective immune response
during influenza infection (17). Administration of MCP-1 also
protects animals from challenge with lethal doses of Salmonella
typhimurium and Pseudomonas aeruginosa (18). Several animal
models with depleted macrophages demonstrate the essential
role of macrophages in controlling influenza virus replication.
Enhanced lethality occurs in macrophage-depleted mice after
infection with genetically reassorted H1N1 virus containing
hemagglutinin (HA) and neuraminidase (NA) of 1918 virus
(19). Depletion of macrophages during H1N1 infection in pigs
causes 40% lethality with decreased antibody titers and CD81
lymphocytes expressing IFN-g (20).
Alveolar macrophages are present in close proximity with
alveolar epithelial cells, and communication between these cells
is important for maintaining homeostasis within the alveoli. The
alveolar lining is covered by alveolar type I and type II
epithelial cells, which play essential roles in fluid balance and
in secretion of surfactant proteins SP-A, SP-B, SP-C, and SP-D
(21–24). Although enhanced lethality in macrophage-depleted
animals is attributed to elevated virus titers, the fate of alveolar
epithelial cells in macrophage-depleted animals after influenza
virus infection is unclear. While previous studies implicate the
Narasaraju, Ng, Phoon, et al.: Roles of Macrophages and HGF in Influenza Pneumonitis
role of macrophages in reducing virus titers, there are no studies
on whether macrophages participate in the protection or resolution of alveolar epithelium. Macrophage lineage cells promote
tissue repair by producing potential growth factors such as
hepatocyte growth factor or HGF (25, 26). HGF is a mitogen
for various types of epithelia, including bronchial and alveolar
epithelial cells (27, 28). Recombinant HGF augments DNA
synthesis of alveolar type II cells in acute lung injury, and also
reduces pulmonary fibrosis during bleomycin-mediated lung injury (29). MCP-1 can induce HGF production in macrophages,
and can also enhance their phagocytotic ability (30).
In this study, we investigated the fate of the alveolar
epithelium in a murine model of influenza pneumonitis after
inhibition of macrophage recruitment into the lungs using anti–
MCP-1 monoclonal antibody treatment. Histopathology, immunohistochemistry, Western blot, reverse transcription-polymerase
chain reaction (RT-PCR), and terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assays
were performed to evaluate the extent of alveolar damage.
HGF production in bronchoalveolar lavage fluid (BALF) and
lung homogenate after infection and anti–MCP-1 treatment was
investigated. In addition, cultured lung explants were treated
with recombinant HGF to characterize the role of HGF in lung
repair after influenza viral infection.
MATERIALS AND METHODS
Mice, Influenza Virus Infection, and Anti–MCP-1 Treatment
Female 4- to 6-week-old BALB/c mice were housed in micro-isolator
cages in an animal BSL-2 laboratory facility. All animal protocols were
approved by the Institutional Animal Care and Use Committee, National University of Singapore. Animals were divided into four groups,
and anesthetized with 375 mg/kg Avertin. Animals were infected intranasally with 30 ml of mouse-adapted human influenza virus A/Aichi/2/
68Influenza virus at 104 TCID50 (6). The anti–MCP-1 treatment group
(n 5 15) received three intraperitoneal injections of 15 mg per dose of
anti–MCP-1 monoclonal antibody (R&D Systems, Minneapolis, MN) at
12, 24, and 48 hours after infection. The control group (n 5 15) received
15 mg per dose of mouse nonimmune immunoglobulin (Ig)G (R&D
Systems) at similar time-points after infection. Another control group
was infected but untreated (n 5 10), while the last control group
received normal uninfected mouse lung homogenate (n 5 10). Animals
were monitored daily for clinical signs of infection, including body
weight loss, and all mice were killed on Day 4.
Determination of Virus Titers
The virus titers in the lung homogenate were assayed by infectivity in
MDCK cells. TCID50 was determined by a reduction in cytopathic
effect (CPE) of 50%, and the log TCID50 of virus per milliliter of
homogenate was calculated.
Bronchoalveolar Lavage Fluid Analyses
For bronchoalveolar lavage fluid (BALF) sample collection, animals
were anesthetized, the trachea was exposed, and the lungs were washed
twice with 0.5 ml of cold phosphate-buffered saline (PBS). The recovery
of the lavage fluid was approximately 90%. The BALF samples were
centrifuged at 1,100 3 g for 10 minutes, and the supernatants were immediately frozen at 2808C until further use. The cell pellets were
resuspended in PBS, and total cell counts were measured using
a hemocytometer. For differential cell counts, the cells were processed
onto microscopic slides using a Cytofuge 2 cytocentrifuge (StatSpin,
Westwood, MA), and subjected to modified Giemsa staining. Cells
(500 per animal) were counted at a magnification of 3400.
Measurement of Mouse Keratinocyte-Derived Chemokine,
Leukotriene B4, and HGF Levels by Enzyme-Linked
Immunosorbent Assay
Serum concentrations of mouse keratinocyte-derived chemokine (KC) and
leukotriene B4 (LTB4) were measured using double-ligand enzyme-linked
733
immunosorbent assay (ELISA) and competitive enzyme immunoassay
(R&D Systems) according to the manufacturer’s instructions. HGF
levels in BALF and macrophage–LA-4 mixed culture supernatant were
determined using a double-ligand ELISA kit (Institute of Immunology,
Tokyo, Japan) according to the manufacturer’s recommendations.
Determination of Myeloperoxidase Enzyme Activity
Myeloperoxidase (MPO) activity in the lung homogenate was assayed
as described previously (31). Briefly, lung homogenate (20 ml) was
mixed with MPO assay solution (980 ml). The latter was prepared fresh
before use by mixing 107.6 ml of H2O, 12 ml of 0.1 M sodium phosphate
buffer (pH 7.0), 0.192 ml of guaiacol, and 0.4 ml of 0.1 M H2O2. The
generation of tetraguaiacol was measured spectrophotometrically at
470 nm wavelength, and the change in optical density (DOD) per
minute was calculated from the initial rate. The MPO activity was then
calculated using the formula (units/ml 5 DOD/min 3 45.1), and
expressed as units per milligram of protein. One unit of the enzyme
is defined as the amount that consumes 1 mmol of H2O2 per minute.
Evaluation of Pathogenicity by Histopathology
All animals from each group were studied by histologic analyses of
their lungs. Lung tissues were fixed in 4% formaldehyde in PBS,
dehydrated, and embedded in paraffin. Paraffin sections of 4 mm
thickness were cut, dewaxed, and stained with hematoxylin-eosin for
histopathologic evaluation under light microscopy. Histopathology
slides were scored in a blinded manner on the basis of the following
parameters: necrotizing bronchiolitis (damage of airway epithelial cells,
presence of necrotic bodies, or denudation of the entire airway lining);
inflammation in bronchioles (bronchioles filled with inflammatory cells,
including macrophages, neutrophils, and lymphocytes); alveolitis (damaged alveolar epithelial cells with denudation or necrosis, and their
presence in the alveoli); interstitial inflammation (inflammation in
alveoli or thickening of alveolar interstitium); hemorrhage (presence of
erythrocytes in the alveolar space due to capillary leakage or endothelial damage); and edema (presence of proteinaceous material in the
alveolar space). The severity of damage was scored on a scale ranging
from 0 to 4 (0 for none or very minor, 1 for mild, 2 for intermediate,
3 for moderately severe, and 4 for severe and widespread). The total
lung surface was scored at 3400 magnification, and each overall score
was expressed as mean value 6 SE.
Western Blot Analysis
Frozen lung tissues were homogenized in lysis buffer (10 mM Tris-HCl
pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 mg/ml
aprotonin, and 10 mg/ml leupeptin), and protein concentration was
determined using the DC protein assay kit (Bio-Rad, Hercules, CA).
Protein (30 mg) was solubilized in SDS sample buffer (62.5 mM Tris-HCl
pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 10% glycerol, 0.01% bromophenol blue), separated on SDS-PAGE (12%), and transferred onto
a nitrocellulose membrane at 100 mA for 2 hours. To check the
efficiency of protein transfer, each membrane was stained with Ponceau
S and blocked for 1 hour with 5% milk in 100 mM Tris-buffered saline
containing 0.1% Tween 20 (TBST). Each membrane was incubated at
48C overnight with 1:1,000 dilutions of anti–SP-C (Santa Cruz Biotechnology, Santa Cruz, CA), anti–T1-a, or anti–b-actin (Sigma, St.
Louis, MO) antibodies. Each membrane was then washed thrice (5 min
each) in TBST, and incubated with horseradish peroxidase–conjugated
anti-mouse or anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology) for
1 hour. After washing thrice (5 min each), each blot was developed with
enhanced chemiluminescence reagents and exposed to X-ray film to
visualize the protein bands. Densitometric analyses were performed
using a Bio-Rad densitometer for all proteins, and the intensity of each
target protein was expressed as a percentage of the b-actin band.
Semi-Quantitative RT-PCR
Total RNA was isolated from frozen lung tissues using the RNeasy
Plus Mini kit (Qiagen, Hilden, Germany), and reverse-transcribed into
cDNA with the MMLV Reverse Transcription system (Promega,
Madison, WI) using random primers (32). Aliquots of cDNA (1 ml
each) were amplified by PCR using SP-C, T1-a, HGF, and b-actin
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
primers (Table 1). The thermal cycling profile was 948C for 3 minutes,
followed by 34 cycles each at 948C for 1 minute, 508C for 1 minute, and
728C for 1 minute, with a final extension of 728C for 10 minutes. For
b-actin, PCR was performed for up to 22 cycles to ensure amplification
in the exponential range. The RT-PCR products from each animal
were quantified using a Bio-Rad densitometer, and each band intensity
was expressed as a percentage of the corresponding b-actin amplicon.
TABLE 1. PRIMERS USED FOR SEMIQUANTITATIVE RT-PCR
Immunohistochemistry
Definition of abbreviations: HGF, hepatocyte growth factor; SP-C, surfactant
protein C.
Lung sections were deparaffinized in xylene, permeabilized with 0.5%
Triton X-100 in PBS for 20 minutes, and blocked with 5% milk in PBS
for 30 minutes. The sections were then incubated at 48C overnight with
1:100 dilutions of primary antibodies (i.e., mouse anti–T1-a, anti–SP-C,
anti-PCNA [Santa Cruz Biotechnology] or anti-HGF [R&D Systems]).
After washing thrice with PBS for 5 minutes, the slides were incubated
with 1:250 dilutions of secondary antibodies conjugated to Alexa 546 or
488 (Molecular Probes, Eugene, OR) at room temperature for 1 hour.
The slides were washed thrice with PBS, mounted, and examined using
an Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan). For
quantitative analysis of type II epithelial hyperplasia, lung sections
were double-stained with anti–SP-C and anti-PCNA. Cells staining
positive for both antigens were considered as proliferating, and the
ratio of double-positive cells to total SP-C–positive cells was ascertained. At least five fields from each lung section at 3400 magnification
were evaluated, and data were obtained from all animals per group.
For detection of HGF, BALF cells were subjected to cytocentriguation, fixed with 4% formaldehyde, permeabilized with 0.5% Triton X100, incubated with anti-HGF antibody (R&D Systems), and mounted
with mounting medium containing diamidino-2-phenylindole or DAPI
(Vector Laboratories, Burlingame, CA).
Detection of Apoptosis by TUNEL Assay
TUNEL assay was performed on 4-mm formalin-fixed slides using the
In Situ Cell Death Detection kit (Roche Diagnostics, Indianapolis, IN)
according to the manufacturer’s instructions (33). For quantification of
alveolar epithelial cells, at least five randomly selected fields at 3400
magnification were selected per lung section in a blinded manner. Cells
on the surface of the alveolar epithelium were counted, but those
within the airspace or not clearly attached to the alveolar surface were
excluded. To calculate the apoptosis index, the number of TUNELpositive alveolar cells was expressed as the percentage of the total
number of nuclei in the same field. Sections from all mice in the
treatment and control groups were analyzed.
Isolation of Mouse Alveolar Macrophages
Animals were anesthetized and alveolar macrophages were harvested by
repeated lung lavage with PBS (34). The lavaged cells were centrifuged
at 1,000 3 g at 48C for 10 minutes, and then resuspended in Dulbecco’s
modified Eagle’s medium. Viable cells were counted by the trypan blue
exclusion assay. Giemsa staining of cytospin preparations showed that
more than 98% of cells were normal lung macrophages.
Preparation of Influenza Virus–Infected LA-4 Cells and In Vitro
Assay for HGF Production
LA-4 mouse lung epithelial cells (American Type Culture Collection,
Manassas, VA) were cultured overnight in F12K medium with 15%
fetal bovine serum. Cells were infected with mouse-adapted influenza
virus (at MOI of 4) in serum-free medium for 1 hour, replaced with the
same medium, and further incubated at 378C for 12 hours. Immunocytochemistry using polyclonal anti-influenza antibody (6) showed that
at MOI of 4, 95% of cells stained positive for influenza viral antigen at
12 hours after infection. To determine whether macrophages produce
HGF when exposed to infected epithelial cells, freshly isolated alveolar
macrophages from mouse lungs were mixed with infected LA-4 cells at
a ratio of 2:1, and incubated for 30 minutes in the presence or absence
of anti–MCP-1 antibody (1 mg/ml). Cells were washed thoroughly to
remove unattached cells (mostly infected LA-4 cells), and further
incubated for 24 hours. Cell culture supernatants were then collected to
measure HGF levels by ELISA. Cells were lysed, total RNA was
extracted, and semiquantitative RT-PCR was performed to determine
HGF transcript levels. The amplified product from each animal was
Gene
SP-C
T1-a
HGF
b-actin
Forward Primer
Reverse Primer
59-AGCAAAGAGGTCCTGATGGA-39
59-GCCAGTGTTGTTCTGGGTTT-39
59-GCCAGGTGACCTTTGCTTTA-39
59-ACTGGGACGACATGGAGAA-39
59-AATCGGACTCGGAACCAGTA-39
59-TGATTCCAACCAGGGTGACT-39
59-TGAACGTAAAGCCCCTGTTC-39
59-TCTCAGCTGTGGTGGTGAA-39
quantified by densitometry, and expressed as the percentage of the
intensity of the corresponding b-actin amplicon.
Phagocytic Activity
Alveolar macrophages from uninfected animals were isolated, counted,
and mixed with uninfected or virus-infected LA-4 cells at a ratio of 1:2.
To assess phagocytic activity, co-cultured cells were incubated at 378C
for 1 hour and washed extensively with PBS to remove unattached cells.
Cells were then fixed in 4% formaldehyde, and permeabilized with
0.05% Triton X-100. Ingested LA-4 cells were detected by staining with
antibody against virus, while macrophages were stained with F4/80
antibody (Santa Cruz Biotechnology). Macrophages displaying viral
immunostaining were considered positive for phagocytosis. Experiments
were performed in duplicate, and a total of five randomly selected fields
per well was observed in triplicate. Phagocytic index was calculated by
measuring the percentage of positive cells out of total cells.
Culture of Lung Explants and Treatment with
Recombinant HGF
Female BALB/c mice were anesthetized, the trachea was exposed, the
lungs were cleared of blood by perfusion with cold PBS and immediately placed in BGJb medium (Gibco, Invitrogen, Carlsbad, CA)
containing penicillin, streptomycin, and neomycin. The lungs were
sliced into 1-mm-thick sections, and each slice (in 4 mg/ml trypsinEDTA) was infected with an influenza virus inoculum of 0.53106
TCID50 for 1 hour, placed on Transwell translucent polycarbonate
culture dish inserts of 8.0 mm pore size, 12 mm diameter (Costar, High
Wycombe, UK), and cultured with serum-free BGJb medium. At
12 hours after infection, the wells were supplemented with 500 ng/ml of
recombinant HGF (R&D Systems) or 10 mg/ml of anti-HGF monoclonal antibody. BrdU (2 mM) was added to the medium 4 hours
before lung explants were collected at 24, 48, and 72 hours after
infection. The explants were fixed with 4% formaldehyde in PBS, and
processed for histologic analysis as described above.
Evaluation of Alveolar Type II Cell Proliferation
Immunohistochemistry of the lung explants was performed to measure
alveolar type II cell proliferation. Lung sections were processed for
immunostaining, and incubated overnight at 48C with anti-mouse BrdU
(1:200), anti–SP-C (1:100), and anti-PCNA (1:100) antibodies. Secondary anti-mouse–Alexa-546 and anti-rabbit–Alexa-488 antibodies were
used for detection, and slides were mounted with DAPI-containing
medium. For quantitative analysis of type II epithelial proliferation,
data were obtained from two independent experiments performed in
triplicate at each time-point, and at least five fields from each section
at 3400 magnification were evaluated.
Statistical Analyses
Each result was expressed as mean 6 SE. Statistical analyses were
performed by ANOVA or Student’s t test. Differences in animal
weights were analyzed by the Mann-Whitney U test. A value of P ,
0.05 was considered statistically significant.
RESULTS
Anti–MCP-1 Treatment Decreases Leukocyte Recruitment to
Infected Lungs
To determine the cellular infiltrates into infected lungs after
anti–MCP-1 treatment, BALF-associated cells were subjected
Narasaraju, Ng, Phoon, et al.: Roles of Macrophages and HGF in Influenza Pneumonitis
to Giemsa staining. Elevated numbers of total leukocytes
including macrophages and neutrophils were noted after influenza virus infection. Infected animals injected with nonimmune IgG also displayed increased cellular infiltration
comparable with the infected group. In contrast, treatment with
anti–MCP-1 antibody significantly reduced infiltration of both
macrophages and neutrophils (Figure 1A).
Effect of Anti–MCP-1 Treatment on Animal Weights
and Virus Titers
Compared with control uninfected mice, the weights of infected
animals treated with anti–MCP-1 revealed a decreasing trend at
all time-points. Significant weight loss was observed especially
on Day 4 (Figure 1B). Animals treated with anti–MCP-1
experienced more than 20% weight loss, and two treated mice
died on Day 4. However, no significant differences in virus titers
were observed among untreated and antibody treatment groups
(Figure 1C).
735
Anti–MCP-1 Treatment Reduces MPO Activity and Serum
Mouse KC Levels
Given that anti–MCP-1 treatment reduced the total number of
neutrophils in BALF, we further investigated whether blocking
MCP-1 influenced the levels of mouse KC, the murine analog of
human IL-8 which is a chemoattractant for neutrophils. Significant reduction of mouse KC in serum was observed (Figure
2A). Congruent with this finding, MPO levels were drastically
reduced in lungs of anti–MCP-1–treated animals compared with
the infected or IgG-treated groups (Figure 2B). The LTB4
levels (in pg/ml) for the four groups were 20.5 6 2.5 (anti–MCP1 treatment), 22.8 6 2.7 (IgG treatment), 21.5 6 3.0 (infected),
and 10 6 1.6 (uninfected).
Histopathologic Analyses Reveal Differences
in Pulmonary Damage
To investigate the histopathologic changes in influenza virusinfected animals after anti–MCP-1 treatment, histopathologic
Figure 1. (A) Effect of anti–monocyte chemoattractant protein (MCP)-1 treatment on leukocyte recruitment into murine lungs after infection with
104 TCID50 of mouse-adapted human influenza A/Aichi/2/68 virus. Lung homogenates (LH) prepared from uninfected mice served as the
uninfected control. Cellular infiltrates were evaluated in bronchoalveolar lavage fluid (BALF) samples collected on Day 4 after infection without and
with treatment with anti–MCP-1 or nonimmune IgG. Total cell counts were significantly increased in virus-infected and nonimmune IgG-treated
groups compared with uninfected animals. In contrast, both macrophage and neutrophil numbers were drastically diminished in the anti–MCP-1–
treated group. Results are expressed as means 6 SE, with n 5 5 per group. *P , 0.05 versus LH group; **P , 0.05 versus infected but untreated group.
(B) Changes in body weights of BALB/c mice after infection and treatment with antibodies. Animal weights were recorded daily for 4 days, and expressed as
means 6 SE, with n 5 10 for LH and virus-infected groups, and n 5 15 for anti–MCP-1 and IgG treatment groups. (C) Determination of virus titers in mouse
lung homogenates. MDCK cells were infected with serial 10-fold dilutions of lung homogenates from uninfected, virus-infected, anti–MCP-1–treated, and
IgG-treated animals. The numbers on the y axis represent virus titers expressed as 10y TCID50 per gram of total protein (means 6 SE), with n 5 5 for the
virus-infected group and n 5 10 for anti–MCP-1 and IgG treatment groups. The virus titers (z 107) of the three infected groups were similar.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
Figure 2. Effect of anti–MCP-1 antibody treatment on levels of (A) serum mouse keratinocyte-derived chemokine (KC) and (B) lung MPO. Both
mouse KC and myeloperoxidase (MPO) levels were significantly decreased in anti–MCP-1–treated infected animals. Data are represented as
means 6 SE, with n 5 5 for uninfected and infected groups, and n 5 10 for anti–MCP-1 and IgG treatment groups. *P , 0.05 versus LH group;
**P , 0.05 versus infected but untreated group.
analyses were evaluated using a scoring system. As shown in
Table 2, significant alveolitis was evident in anti–MCP-1–treated
animals compared with IgG-treated or nontreated infected
mice. The alveolar spaces exhibited enlargement, with some
denuded areas showing complete loss of epithelium. However,
cellular infiltration was more prominent in the untreated group
compared with the anti–MCP-1–treated group. Although alveolar epithelial damage was observed in both untreated and IgGtreated animals, hyperplasia of type II alveolar epithelial cells
was also noted (Figure 3). Interstitial inflammation was less
conspicuous in anti–MCP-1–treated and IgG-treated animals
compared with untreated controls. No significant difference in
inflammation in bronchioles, hemorrhage, or edema was noted
among untreated and antibody-treated infected animals.
Pronounced Alveolar Epithelial Damage and Apoptosis after
Anti–MCP-1 Treatment
The alveolar epithelium is a major target for influenza virus
replication, which was confirmed by histopathologic evidence of
remarkable alveolitis. To further evaluate the extent of alveolar
epithelial damage, we specifically investigated T1-a as well as
SP-C, which represent surface epithelial markers for alveolar
type I and type II pneumocytes, respectively. Our results
revealed considerable reduction in expression of these markers
at both protein (Figures 4A and 4B) and transcriptional levels
(Figures 4C and 4D) in anti–MCP-1–treated animals. Immunostaining for SP-C protein showed drastic decrease in SP-C
staining intensities in anti–MCP-1–treated animals compared
with the other three control groups. Staining for T1-a was continuous in the lungs of control uninfected mice. Despite enhanced cellular infiltration in untreated and IgG-treated infected
animals, T1-a staining was intense. In contrast, the lungs of anti–
MCP-1–treated animals displayed discontinuous T1-a staining,
indicating type I pneumocyte damage (Figure 4E). Alveolar type
II epithelial hyperplasia was also determined by double immunostaining for PCNA and SP-C (Figure 5). The percentages of
cells positive for both PCNA and SP-C were significantly
elevated in untreated and IgG-treated infected animals, thus
indicating alveolar type II epithelial proliferation.
By the TUNEL assay, animals treated with anti–MCP-1
demonstrated increase in apoptotic cells in the alveolar septae
(Figures 6A and 6B). Although the total numbers of apoptotic
alveolar epithelial cells in untreated and IgG-treated infected
animals were comparatively fewer, their lungs displayed more
apoptotic cells within the alveolar spaces, the majority of which
were macrophages with engulfed infected cells. In contrast,
alveolar spaces of anti–MCP-1–treated animals exhibited fewer
infiltrated cells showing apoptosis.
Enhanced HGF Production in BALF and Lungs after Influenza
Virus Infection
The recovery of BALF from all animals was approximately
90%, and HGF levels in BALF were measured by ELISA. HGF
protein levels were significantly increased in untreated compared with anti–MCP-1–treated infected animals (Figure 7A).
In agreement with this, HGF mRNA expression in lung tissue
was significantly higher in control versus anti–MCP-1–treated
animals (Figures 7B and 7C). Both HGF protein and mRNA
levels were similar in the untreated and IgG-treated groups.
Immunostaining for HGF in lung tissue and BALF cells
revealed strongly positive signals mainly in alveolar macrophages, with occasional staining in alveolar epithelial cells.
BALF cells also displayed HGF staining only in macrophages
but not in neutrophils or lymphocytes (Figure 7D).
TABLE 2. SEMIQUANTITATIVE SCORING OF LUNG HISTOPATHOLOGY
Sample
LH
Virus
Virus 1 MCP-1
Virus 1 IgG
Necrotizing
Bronchiolitis
0.2
1.2
1.9
2
6
6
6
6
0.4
1.1
1.0
0.7
Inflammation in
Bronchioles
1
2.2
1.9
2.4
6
6
6
6
0.7
1.0
0.9
0.8
Alveolitis
0
1.2 6 0.8
2.5 6 0.7*
1.6 6 0.8
Interstitial
Inflammation
0.5
3
1.7
2.2
6
6
6
6
0.5
0.9†
0.9
1.0
Hemorrhage
Edema
0
0.8 6 0.9
0.8 6 0.8
0.6 6 0.6
0
1.1 6 0.9
0.7 6 0.7
0.7 6 0.6
Definition of abbreviations: IgG, immunoglobulin G; LH, lung homogenates; MCP-1, monocyte chemoattractant protein-1.
* P , 0.05 versus virus group and virus 1 IgG group.
†
P , 0.05 versus virus 1 MCP-1 group.
Narasaraju, Ng, Phoon, et al.: Roles of Macrophages and HGF in Influenza Pneumonitis
737
Figure 3. Effect of anti–MCP-1 treatment on histopathologic
changes in lungs at Day 4 after infection. Animals administered with uninfected LH displayed normal architecture of
airway (BR) and alveolar epithelia (AV) without inflammation
(A, B). Mice infected with influenza virus (C, D) and treated
with IgG (G, H) exhibited increased cellular infiltration with
mild damage of both airway and alveolar epithelia. Prominent hyperplasia of alveolar epithelial cells was also observed
in both infected and IgG-treated animals (arrows). Enhanced
alveolar and airway epithelial damage with enlarged airspaces
and some denuded epithelial lining (arrowheads) were noted
in infected animals treated with anti–MCP-1 (E, F). Tissue
sections were stained with hematoxylin and eosin, and examined at magnifications of 3100 and 3400 in the left and right
columns, respectively. Semiquantitative histopathology scoring
was performed (n 5 5 for LH and infected groups and n 5 10
for anti–MCP-1 and IgG treatment groups), with the scores
shown in Table 2.
Co-Culture of Infected Cells with Alveolar Macrophages
Augments the Production of HGF
was not significantly altered in the presence of anti–MCP-1
antibody.
To investigate whether macrophages produce HGF when exposed to influenza virus–infected epithelial cells, we conducted
in vitro co-culture studies using freshly isolated mouse alveolar
macrophages with influenza virus–infected LA-4 cells. HGF
levels in culture supernatants were significantly elevated when
macrophages were incubated with infected LA-4 cells compared
with uninfected cells (Figure 8A). Accordingly, HGF mRNA
expression was also induced in macrophages co-cultured with
infected epithelial cells (Figures 8B and 8C). Further addition of
anti–MCP-1 to the co-culture did not inhibit HGF production.
Uninfected macrophages and LA-4 cells alone or in co-culture
showed no induction of HGF expression.
Treatment with Recombinant HGF Stimulates Alveolar
Epithelial Cell Proliferation in Murine Lung Explants
Anti–MCP-1 Treatment Does Not Affect the Phagocytic
Activity of Alveolar Macrophages
To explore whether MCP-1 plays a role in the phagocytosis of
influenza virus–infected cells, freshly isolated macrophages
were incubated with uninfected or infected LA-4 cells in
the presence or absence of anti–MCP-1 (1 mg/ml). Cells were
then washed and stained with anti-influenza and anti-F4/80
antibodies. An increase in phagocytic index (22.6 6 0.7) was
observed when macrophages were incubated with infected
epithelial cells. However, phagocytic activity (24.9 6 1.2)
To interrogate the role of HGF in alveolar epithelial repair, lung
explants were treated with recombinant HGF or anti-HGF monoclonal antibody after infection. BrdU labeling assay exhibited
significantly higher numbers of alveolar type II epithelial cells
positive for both BrdU and SP-C in recombinant HGF-treated
lung explants compared with anti-HGF–treated or untreated infected cultures (Figure 9). Alveolar type II epithelial cell proliferation was approximately 2.7-fold higher in HGF-treated
infected explants compared with uninfected controls at 48 and
72 hours after infection. These findings clearly indicate the involvement of HGF in the mechanism of alveolar epithelial regeneration during influenza virus infection.
DISCUSSION
Recent studies indicate that alveolar macrophages may play
a protective role during influenza virus infection. Although
cytokines produced by macrophages are implicated in the pathogenesis of severe influenza virus infections (11–13), macrophagedepleted animals infected with influenza virus suffer high
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
Figure 4. Evaluation of alveolar epithelial damage by Western blot, RT-PCR,and immunohistochemistry. Alveolar epithelial markers including T1-a
(for type I epithelium) and surfactant protein (SP)-C (for type II epithelium) were used to evaluate alveolar epithelial damage. (A) Western blot
analyses depicting T1-a and SP-C expression in lung homogenates of (1) uninfected, (2) virus-infected, (3) anti–MCP-1, and (4) IgG treatment
groups. Anti–MCP-1–treated animals showed significantly reduced expression of SP-C and T1-a, with b-actin (42 kD) serving as a loading control.
(B) Densitometric analyses of Western blot bands, each expressed as a percentage of the corresponding control b-actin band (with the LH group
assigned as 100%). (C) Semiquantitative RT-PCR assay for mRNA expression of T1-a and SP-C. (D) Densitometric analyses of RT-PCR amplicons,
each expressed as a percentage of the corresponding control b-actin amplicon (with the LH group assigned as 100%). Representative results are
shown for uninfected and infected groups (n 5 5 each), and for anti–MCP-1 and IgG treatment groups (n 5 10 each). Asterisk denotes statistical
significance at P , 0.05 versus LH group. (E) Immunostaining for detection of T1-a and SP-C in lung sections counterstained with DAPI nuclear dye.
Uninfected lung section shows continuous T1-a staining (arrows) covering 95% of the alveoli. Strong SP-C signals (arrowheads) are depicted in
uninfected and IgG-treated groups. However, discontinuous T1-a staining (*) and minimal SP-C staining (white arrow) were observed in anti–MCP1–treated animals.
Narasaraju, Ng, Phoon, et al.: Roles of Macrophages and HGF in Influenza Pneumonitis
Figure 5. Quantitative analysis for alveolar type II cell hyperplasia by
staining lung sections with SP-C and PCNA. Out of the total SP-C–
positive cells, the number and percentage of cells positive for both
PCNA and SP-C were determined for each group. Results are expressed
as mean percentages 6 SE, with n 5 5 for uninfected and infected
groups, and n 5 10 for anti–MCP-1 and IgG treatment groups. *P ,
0.05 versus LH group; **P , 0.05 versus infected group.
mortalities compared with nondepleted controls (19, 20). Alveolar macrophages exist in close proximity with alveolar epithelium, which is a major target for influenza virus replication.
Although elevated viral titers are thought to contribute to
enhanced lethality in macrophage-depleted animals, the fate
of the alveolar epithelium is unclear. In this study, we provide
evidence of enhanced alveolar epithelial damage and apoptosis
after blocking macrophage recruitment by treatment with anti–
MCP-1 antibody in a mouse model of influenza pneumonitis.
Control mice sub-lethally infected with influenza virus, and
control infected mice treated with nonimmune IgG, displayed
hyperplasia of alveolar type II cells and enhanced HGF production in BALF. In contrast, no epithelial proliferation and
decreased HGF levels were observed in anti–MCP-1–treated
739
infected animals. Furthermore, HGF expression was induced in
alveolar macrophages incubated with influenza virus–infected
LA-4 cells, and recombinant HGF treatment of infected lung
explants stimulated alveolar type II epithelial cell proliferation,
thus suggesting the potential role of macrophages in the
resolution of alveolar epithelium.
MCP-1 belongs to the family of CC chemokines with pleiotropic activities. It is produced by many different cell types, and is
a major chemoattractant for leukocytes. Hyperoxia-mediated
lung injury and septic peritonitis models reveal that treatment
with anti–MCP-1 antibody reduces leukocyte infiltration in the
lungs and peritoneum, respectively (35, 36). Here we provide
evidence that anti–MCP-1 treatment significantly reduced infiltration of macrophages into the lungs after influenza virus
infection. Given that elevated MCP-1 levels were detected by 12
hours (data not shown), anti–MCP-1 antibody treatment was
commenced 12 hours after infection and continued until 48 hours
to achieve significant reduction in macrophage infiltration. Blocking MCP-1 also reduced the neutrophil population and MPO level
in bronchoalveolar lavage. Similarly, mice lacking the MCP-1 gene
display diminished pulmonary infiltration of both macrophages
and neutrophils after influenza virus infection (17). Moreover,
after anti–MCP-1 treatment, reduction of serum murine KC (a
chemokine similar to cytokine-induced neutrophil chemoattractant-1 or CINC-1), but not of the lipid mediator leukotriene B4,
suggest that mouse KC may influence the recruitment of neutrophils during influenza virus infection. These results concur with
previous studies elucidating the role of mouse KC and MIP-2 in
neutrophil recruitment in influenza virus infection (37). In addition, MCP-1 facilitates the pulmonary recruitment of CD81 T cells
that are crucial in host defense against viral infection (20). Hence,
reduced MCP-1–dependent CD81 T cell recruitment may also
contribute to the observed pathologic effects.
Compared with infected but untreated control animals,
infected mice treated with anti–MCP-1 experienced significant
weight loss on Day 4 after infection, with two animals dying
on Day 4. These observations are compatible with studies on
macrophage depletion in pigs and mice that report increased
mortality upon challenge with sub-lethal influenza virus doses.
Figure 6. Detection of apoptosis by TUNEL assay. (A) Representative micrographs depicting lungs of (i) uninfected, (ii) virus-infected, (iii) anti–MCP-1
treatment, and (iv) IgG treatment groups. Arrows indicate apoptotic epithelial cells, while arrowheads show TUNEL-positive macrophages at 3400
magnification. (B) Quantification of apoptosis was determined by the number of positively stained nuclei within the alveolar epithelial lining. The apoptosis
index represents the average percentage of TUNEL-positive alveolar epithelial cells, and the results are expressed as means 6 SE, with n 5 5 for LH and
infected groups, and n 5 10 for anti–MCP-1 and IgG treatment groups. *P , 0.05 versus LH group; **P , 0.05 versus infected but untreated group.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
Figure 7. Effect of anti–MCP-1 treatment on HGF production in BALF and lungs. (A) HGF levels in BALF measured by ELISA (n 5 5 per group). (B)
Expression of HGF mRNA in lungs analyzed by RT-PCR exemplifying HGF-specific fragments amplified from total RNAs of lungs of three
representative mice per group. (C) Densitometry analysis, with each HGF RT-PCR amplicon expressed as a percentage of the corresponding b-actin
control amplicon. Data are represented as means 6 SE, with n 5 5 for uninfected and infected groups, and n 5 10 for anti–MCP-1 and IgG
treatment groups. *P , 0.05 versus LH; **P , 0.05 versus infected group. (D) Immunostaining for HGF detection in lung sections and BALF cells
counterstained with DAPI nuclear dye. (i) Uninfected lung section shows occasional staining in alveolar epithelial cells (arrowheads). Strong HGF
signals are apparent in macrophages (arrows) of (ii) infected lung sections and (iii) BALF cells. (iv) Distinct HGF staining in macrophages (arrows) but
absent in neutrophils (open arrows) in BALF.
Depletion of macrophages in mice before infection with
recombinant influenza virus containing the HA and NA genes
of highly virulent 1918 virus (1918 HA/NA:TX91 strain) leads
to decreased levels of cytokines (including TNF-a, IFN-b,
IFN-g, MIP-1a, and MIP-2), but culminates in elevated viral
load, systemic viral spread into the brain, and enhanced
lethality. However, depletion of macrophages 3 days after
infection does not have any impact on the disease outcome.
Similar findings with reduced TNF-a and IFN-g, increased
viral load and lethality are noted in another study of macrophage-depleted pigs infected with human H1N1 virus (A/New
Caledonia/20/99). Notwithstanding that previous studies document enhanced virus titers in macrophage-depleted animals
(19, 20), we observed no significant change in virus titers after
anti–MCP-1 treatment. This may be explained by initial
clearance of the virus by resident macrophages, since anti–
MCP-1 treatment commenced only at 12 hours after infection,
whereas macrophages were depleted before infection in other
studies.
We previously demonstrated that influenza virus replicates
efficiently in both alveolar type I and type II epithelial cells,
with the severity of infection being concomitant with loss of
these cells (6). Depletion of macrophages may further enhance
the susceptibility of alveolar epithelium to virus infection. Our
results clearly indicate enhanced alveolar wall disruption
with enlarged alveolar spaces in anti–MCP-1–treated animals.
Decreased lung expression patterns of SP-C and T1-a at both
protein and mRNA levels in anti–MCP-1–treated animals imply augmented susceptibility of epithelium to virus infection in
the absence of macrophages. Congruent with these findings,
significantly more pronounced apoptosis of alveolar epithelium
was observed in anti–MCP-1–treated animals. Although macrophages and neutrophils constitute part of the innate immune
system and act as the first-line defense against influenza virus,
Narasaraju, Ng, Phoon, et al.: Roles of Macrophages and HGF in Influenza Pneumonitis
741
Figure 8. HGF expression in freshly isolated mouse alveolar macrophages incubated with influenza virus-infected LA-4 cells. (A) HGF levels in the
cell culture supernatants were determined by ELISA. (B) RT-PCR analysis of HGF mRNA expression. HGF-specific fragments (upper panel) together
with b-actin fragments (lower panel) were amplified from total RNAs of lungs and subjected to agarose gel electrophoresis. Lanes represent (1)
macrophages, (2) LA-4 cells, (3) macrophages and uninfected LA-4 cells, (4) macrophages and LA-4 cells at 24 hours after infection, and (5)
macrophages and infected LA-4 cells treated with anti–MCP-1 antibody. (C) Amplicons were subjected to densitometry, and the HGF readings
expressed as percentages of the corresponding b-actin amplicons. Data are represented as means 6 SE of two independent experiments. *P , 0.05
compared with cultured macrophages.
alveolar epithelial cells also produce soluble factors such as
surfactant proteins that play important roles in host defense.
Thus, SP-A, SP-B, and SP-D are generated by alveolar type II
and airway epithelial cells, while SP-C is exclusively synthesized
by type II cells. Also known as collectins, SP-A and SP-D
facilitate the elimination of invading pathogens by enhancing
phagocytic activity of macrophages (23, 38, 39). SP-A–deficient
mice exhibit an exaggerated inflammatory response to influenza
virus infection (40). Type I epithelial cells also mediate in
defense by producing apolipoprotein E and transferrin, which
reduce oxidative stress (41). Our data offer strong evidence that
enhanced alveolar epithelial damage can contribute to influenza
pathogenesis after interruption of macrophage recruitment. The
loss of alveolar epithelium causes failure of gas exchange, fluid
imbalance, and inadequate respiration, thereby ultimately culminating in death. Our results concur with studies documenting
enhanced alveolar epithelial apoptosis in autopsy samples of
patients who succumbed to H5N1 infection (42, 43). The
mechanism of lung epithelial apoptosis in influenza pneumonia
has been attributed to macrophage-expressed TNF-related
apoptosis-inducing ligand or TRAIL (44).
Interestingly, in control infected and IgG-treated animals,
apoptosis of alveolar epithelium was observed concomitantly
with hyperplasia of alveolar type II cells. The latter cells are
considered to be pulmonary stem cells involved in the lung
repair process for normal alveolar epithelial regeneration after
injury. Macrophage lineage cells produce HGF, which is
a mitogen for alveolar type II cells (27, 28). Anti–MCP-1
treatment aggravates lung injury in a pneumonia model,
whereas treatment with MCP-1 reduces lung injury due to
enhanced HGF production by macrophages. We next asked
whether HGF is induced during influenza virus infection. The
elevation of HGF protein levels in BALF and of HGF
transcripts in the lungs of control infected and nonimmune
IgG-treated animals, which displayed epithelial cellular hyperplasia, implies the potential role of HGF in epithelial proliferation. Alveolar macrophages exhibited strongly positive
staining for HGF, indicating that these cells are a major source
of HGF production in the lungs after influenza infection.
Furthermore, anti–MCP-1 treatment resulted in significantly
diminished HGF levels, further affirming that macrophages are
important producers of HGF. In concordance with these
findings, we observed significant induction of HGF levels in
alveolar macrophages together with their enhanced phagocytic
activity when co-cultured with virus-infected LA-4 cells. Addition of MCP-1 induces HGF production by macrophages incubated with aged neutrophils. MCP-1 treatment stimulates
HGF production, thus promoting tissue repair during acute
bacterial pneumonia (45). In contrast, MCP-1–deficient mice
show delayed re-epithelialization and collagen synthesis (46).
However, our in vitro study revealed that anti–MCP-1 treatment did not affect HGF secretion and phagocytic activity by
alveolar macrophages during interaction with infected cells,
indicating that there may be other factors involved in stimulating HGF production by macrophages. Lung explants cultured in
the presence of recombinant HGF elicited mitogenic activity of
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
Figure 9. Analysis of alveolar type II epithelial
proliferation in murine lung explants exposed to
recombinant mouse HGF or anti-HGF monoclonal
antibody after influenza virus infection. (A) Immunostaining for detection of SP-C and BrdU in lung
sections (at 3400 magnification) of (i) uninfected,
(ii) infected, (iii) infected and recombinant HGFtreated, and (iv) infected and anti-HGF–treated
lung explants. Double staining (arrows) represent
alveolar type II cells undergoing proliferation. BrdUpositive cells were also observed in bronchiolar
epithelium of HGF-treated lung explants (open
arrow). (B) Quantification of type II epithelial cell
proliferation by staining sections of lung explants
with SP-C and BrdU. Out of the total SP-C–positive
cells, the number and percentage of cells positive
for both BrdU and SP-C were determined. Two
independent experiments were performed in triplicate at each time-point, and results are expressed
as mean percentages 6 SE. *P , 0.05 versus
uninfected.
alveolar type II epithelial cells compared with explants treated
with anti-HGF. Increased BrdU-positive labeling index indicative of DNA synthesis was observed within 24 hours, but more
significant stimulation was observed at 48 and 72 hours. These
results concur with previous reports documenting the mitogenic
effect of HGF in alveolar type II epithelial cells (27, 28).
Moreover, HGF is known to possess antifibrotic activity via
the up-regulation of Smad7 expression in epithelial cells (47).
The addition of anti-HGF blocks the endogenous HGF produced by alveolar macrophages, implying that macrophages are
major producers of HGF in the lung. These findings indicate the
active role of HGF in the regeneration and resolution of
alveolar epithelium after influenza virus infection. Interestingly,
Clara cells of the airway epithelium expressing CC10 (Clara
cell–specific antigen) also exhibited strongly positive BrdU
staining (data not shown), which warrants further investigation.
In conclusion, our study demonstrated that inhibition of macrophage recruitment augmented alveolar epithelial damage and
apoptosis in influenza virus infection, suggesting the protective
role of alveolar macrophages in the resolution of alveolar
epithelium via HGF production. Type II pneumocytes serve
as local stem cells to regenerate damaged alveolar epithelium
via a mechanism that is partially dependent on MCP-1 signaling,
which is involved in pulmonary epithelial repair processes.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
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